Methods and Apparatus for the Location and Concentration of Polar Analytes Using an Alternating Electric Field

ABSTRACT

A method is disclosed for effecting the concentration of a polar analyte in an alternating electric field. In the method, a relative translation of the polar analyte and an alternating electric field along a translation path is effected. A portion of the polar analyte is then trapped and concentrated in a concentration zone formed by the intersection of the translation path and the alternating electric field. Also disclosed are various devices for carrying out the forgoing method.

FIELD OF THE INVENTION

This invention relates to methods and apparatus for the location andconcentration of polar analytes using an alternating electric field.

BACKGROUND

A recent trend in the field of analytical instrumentation has been thedevelopment of integrated microfluidic devices in which multipleoperations are performed on a single device, e.g., Harrison et al.,“Micromachining a Miniaturized Capillary Electrophoresis-Based ChemicalAnalysis System on a Chip,” Science, 261: 895 (1992). Such devices offermany advantages over conventional analytical formats including theability to handle very small volumes; ease and economy of devicefabrication; the ability to integrate multiple operations onto a singleintegrated device; and the opportunity to achieve a high degree ofautomation.

In many chemical and biochemical analysis methods performed usingmicrofluidic devices, it is advantageous to concentrate an analyte aspart of the analysis. For example, increased analyte concentrationgenerally leads to increased chemical reaction rates, increased rates ofmass transfer, and enhanced detectability. However, because conventionalconcentration methods require a solid phase pullout step (e.g.,adsorption), or a phase change of the analyte (e.g., precipitation), ora phase change of the solvent (e.g., evaporation), these methods are notwell adapted for use in a microfluidic device.

In addition, methods for controlling the location of an analyte areimportant in the design of methods using microfluidic devices. Forexample, prior to a separation step, it may be desirable to locate asample volume in a spatially-defined injection zone.

Therefore, it would be desirable to have a method for the location andconcentration of an analyte that is well suited for use in integratedmicrofluidic systems.

SUMMARY

The present invention is directed towards our discovery of methods anddevices for the location and concentration of polar analytes using analternating electric field.

In a first aspect, the present invention provides a method for theconcentration of a polar analyte comprising the steps of effecting therelative translation of the polar analyte and an alternating electricfield along a translation path such that a portion of the polar analyteis trapped and concentrated in a concentration zone formed by theintersection of the translation path and the alternating electric field.

In a second aspect, the present invention provides a device for theconcentration of a polar analyte comprising a translation path, a firstset of electrodes located to provide a first electric field effective tocause the electrokinetic translation of a polar analyte along thetranslation path, and a second set of electrodes located to provide analternating second electric field intersecting the translation path andsufficient to trap and concentrate a portion of the polar analyte in aconcentration zone formed by the intersection of the translation pathand the alternating second electric field.

It is a first object of the invention to provide a method for theconcentration of a polar analyte that does not require a solid-phasepull-out step or a phase change of the polar analyte or a solvent.

It is a second object of the invention to provide a method for themanipulation or location of a polar analyte that does not require asolid-phase pull-out step or a phase change of the polar analyte or asolvent.

It is a third object of the invention to provide a method for theconcentration or manipulation of a polar analyte that does not requiremanual intervention and is therefore well suited to automation.

It is a fourth object of the invention to provide a method for theconcentration or manipulation of a polar analyte that is well suited foruse in a microfluidic device.

The present invention will become better understood with reference tothe following written description, drawings, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a preferred method according tothe present invention.

FIG. 2A is a schematic representation of a preferred microfluidicconcentration and detection device according to the present invention.

FIG. 2B is a schematic representation of a preferred microfluidicconcentration, hybridization, and detection device according to thepresent invention.

FIG. 2C is a schematic representation of a preferred microfluidicconcentration device including a frit located in the concentration zone.

FIG. 3A is a schematic representation of a preferred microfluidicconcentration and electrokinetic separation device according to thepresent invention.

FIG. 3B is a schematic representation of an alternative preferredmicrofluidic concentration and electrokinetic separation deviceaccording to the present invention.

FIG. 4 is a schematic representation of several exemplary electrodeconfigurations adapted for use in the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to certain preferred embodiments ofthe present invention, examples of which are illustrated in theaccompanying drawings. While the invention will be described inconjunction with selected preferred embodiments, it will be understoodthat these embodiments are not intended to in any way limit the scope ofthe invention. On the contrary, the invention is intended to coveralternatives, modifications, and equivalents, which may be includedwithin the scope of the invention as determined by the appended claims.In addition, as used in this disclosure, the plural and singular numberswill each be deemed to include the other; “or” is not exclusive; and“includes” and “including” are not limiting.

Generally, the present invention comprises methods and apparatus forlocating and concentrating a polar analyte at a specified location byeffecting the relative translation of the polar analyte with respect toan alternating electric field along a translation path such that thepolar analyte is concentrated in a concentration zone formed by theintersection of the translation path and the alternating electric field.

The invention is based in part on the discovery that when a polaranalyte passes into an alternating electric field having the appropriatecharacteristics, the polar analyte will become trapped in aconcentration zone formed by the alternating electric field. While thetheoretical explanation for this highly unexpected and beneficialphenomena is not well understood, and the present invention is notintended to be limited in any way by the following or any othertheoretical explanation, it is thought by the inventors that thistrapping phenomenon is related to the Winslow effect, also referred toas the electrorheological effect, in which polar entities alignthemselves head-to-tail along the field lines of an electric fieldthereby forming filamentous structures, e.g., Winslow, J. AppliedPhysics (1947); and Winslow, U.S. Pat. No. 2,417,850.

I. Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

“Translation path” means a path described as a result of a relativetranslation of an alternating electric field and a medium potentiallycontaining a polar analyte. The translation path may have any shape,e.g., straight, curved, and the like.

“Separation channel” means a channel used to conduct a separationprocess, e.g., separations based on electrokinetic, chromatographic, orother like process.

“Electrokinetic translation” means the movement of an entity in responseto an electric field. Exemplary electrokinetic translation processesinclude but are not limited to electrophoresis, dielectrophoresis,electroendoosmosis, micellar electrokinetic chromatography,isotachophoresis, and combinations of the foregoing processes.

“Electrokinetic translation system” means an apparatus effective tocause the electrokinetic translation of a polar analyte. Typically, anelectrokinetic translation system will include a channel for supportinga medium, a power supply, a set of two or more electrodes in electricalcommunication with the channel, and electrical connections between thepower supply and the two or more electrodes.

“Dipole moment” means the product q*R where q and -q are charges ofopposite polarity that are separated by a distance R. As used in thefollowing discussion, a dipole moment includes a permanent dipolemoment, a dipole moment resulting from the electric-field-inducedpolarization of an entity having a finite polarizability, a dipolemoment resulting from the orientation of an entity, e.g., orientationpolarization, a dipole moment resulting from the electric-field-inducedspace-charge distortion of a counter-ion atmosphere surrounding anentity, e.g., a Debye layer, or a dipole moment resulting from anycombination of the foregoing mechanisms.

“Alternating electric field” means an electric field characterized by avector whose magnitude or direction varies with time. Included withinthe definition of an alternating electric field is a multi-phaseelectric field. The limits of the spatial extent of an alternatingelectric field is that region in which the electric field strength ofthe alternating electric field is greater than that required to trap andconcentrate a polar analyte of interest with respect to the alternatingelectric field.

“Polar analyte” means an analyte having a dipole moment.

II. Methods

Referring to FIG. 1, in a preferred embodiment, the method of theinvention comprises a method for the location and concentration of apolar analyte 5 comprising the steps of effecting the relativetranslation of the polar analyte 5 along a translation path 10, andeffecting an alternating electric field 20 which intersects thetranslation path 10 such that some or all of the polar analyte 5 istrapped and concentrated in a concentration zone 30 formed by theintersection of the translation path 10 and the alternating electricfield 20.

The polar analyte 5 for use in the methods of the present invention maybe any analyte having a dipole moment. The polar analyte may be chargedor uncharged, and if the polar analyte is charged, it may have anoverall net charge or be neutral. Preferably, the polar analyte will bepresent in a buffered electrolyte solution, more preferably anelectrolyte solution having a low ionic strength. Exemplary polaranalytes include nucleic acids, both single and double stranded,proteins, carbohydrates, viruses, cells, organelles, organic polymers,particles, and the like. A particularly preferred polar analyte for usein the methods of the present invention is single or double strandednucleic acid dissolved in an electrolyte.

The means used to effect the relative translation of the polar analyte 5with respect to the alternating electric field 20 along the translationpath 10 may be any of a number of different means, or combinations ofmeans, capable of causing the polar analyte to become located in aconcentration zone 30. Such relative translation may be effected bymovement of the polar analyte 5, movement of the alternating electricfield 20, or by movement of both the polar analyte 5 and the alternatingelectric field 20.

For example, the means used to effect the relative translation of thepolar analyte with respect to the alternating electric field may besimple gravity forces. Alternatively, translation of the polar analyte 5may be induced by spinning the system 12 about a selected axis so as toimpose a centrifugal force having a component directed along thetranslation path 10. Instead, the polar analyte 5 may be caused to movealong the translation path 10 by capillary action or othersurface-mediate processes. Alternatively, active hydraulic pumping maybe employed to move the polar analyte 5 along the translation path as aresult of a pressure gradient, e.g., using a conventional microvolumesyringe pump. In yet another preferred embodiment where the polaranalyte is magnetic, a magnetic field may be used to cause thetranslation of the polar analyte along the translation path, e.g., usinga permanent magnet or an electromagnet. In an additional preferredembodiment of the subject invention, an electric field may be used toeffect the translation of the polar analyte by an electrokinetictranslation processes.

Where the means for effecting the relative translation of the polaranalyte with respect to the alternating electric field is effected bymovement of the alternating electric field 20, such movement may beachieved by mounting the electrodes used to form the alternatingelectric field on a moveable stage connected to a conventionalmechanical translation system. The mechanical translation system mayeffect the linear or non-linear translation of the electrodes. Suchmechanical translation systems are well known in the art, e.g.,Hunkapiller et al., U.S. Pat. No. 4,811,218; and Hueton et al., U.S.Pat. No. 5,459,325. Exemplary mechanical translation systems includeelectromechanical systems, e.g., a lead screw or belt drive connected toa motor, piezoelectric actuators, or pneumatic or hydraulic systems,e.g., a piston-in-cylinder drive system. One particularly preferredmechanical translation system comprises acomputer-controlled-DC-servo-motor-driven XY translation stage.

Regardless of the means used to effect the relative translation of thepolar analyte with respect to the alternating electric field along thetranslation path, during the concentration process, the forces used toeffect the relative translation must not be so strong so as to overwhelmthe trapping forces of the alternating electric field. Rather, theforces used to cause the relative translation along the translation pathshould be balanced against the trapping forces used to trap the polaranalyte in the concentration zone as discussed in more detail below.

The alternating electric field 20 used to trap the polar analyte can beany alternating electric field effective to trap and concentrate a polaranalyte. Generally, the alternating electric field of the invention maybe characterized by a time vs. field strength profile, a frequency, anda maximum field strength. The properties of the alternating electricfield required to trap the polar analyte will depend on a number ofeasily accessible experimental parameters including the magnitude of thedipole moment of the polar analyte, the dielectric constant of thesupporting medium, and, in the case of a polar analyte having an induceddipole moment, the polarizability of the polar analyte or surroundingcounterion atmosphere.

The time vs. field strength profile of the alternating electric fieldmay be sinusoidal, sawtooth, rectangular, superpositions of theforegoing, periodic or non-periodic, or any other profile capable ofbeing generated using a modern function generator, e.g., a Model 33120A15 MHz Function/Arbitrary Waveform Generator from Agilent Technologies.Preferably, the time vs. field strength profile of the alternatingelectric field is rectangular. The rectangular profile is preferredbecause it has essentially no zero-field component, resulting in a higheffective duty cycle. In a particularly preferred embodiment, the timevs. field strength profile of the alternating electric field is suchthat the time-averaged integrated field strength is zero where theaverage is taken over one complete cycle. This profile is preferredbecause it minimizes the extent to which a polar analyte located in theconcentration zone will be translated within the concentration zone in adirection other than along the translation path, and it reduces theamount of electrochemical reaction products produced at the surface ofthe electrodes, e.g., gas bubbles.

The frequency of the alternating electric field may be any frequencycapable of trapping a portion of a polar analyte. However, for manypolar analytes of practical importance, the frequency of the alternatingelectric field is preferably between about 10 Hz and 100 megahertz(MHz), and more preferably between about 1 kilohertz (KHz) and 100 KHz.

While the maximum field strength of the alternating electric field maybe any field strength suitable to a particular application, preferably,the maximum field strength of the alternating electric field, asmeasured by the peak field strength of the alternating electric field,is between about 100 V/cm and 10,000 V/cm, and more preferably betweenabout 1,000 V/cm and 20,000 V/cm.

The alternating electric field may be spatially uniform or spatiallynon-uniform. For example, the alternating electric field may be used toeffect dielectrophoretic transport.

A trapped polar analyte may be released from a concentration zone byeither reducing the trapping strength of the alternating electric fieldor by increasing the force used to effect the relative translation ofthe polar analyte with respect to the alternating electric field. Thetrapping strength of the alternating electric field may be modulated bychanging the frequency, field strength, or both. Alternatively, thepolar analyte can be released from the concentration zone by increasingthe forces used to effect the relative translation of the polar analyteand the alternating electric field, e.g., by increasing the electricfield used to drive electrokinetic translation of the polar analyte,increasing the pressure used to drive a pressure-driven flow of thepolar analyte, or increasing the rate of translation of the alternatingelectric field.

In a preferred embodiment, subsequent to an analyte concentration stepaccording to the methods of the present invention, a further analyticalstep is performed. In one preferred embodiment, after a polar analytehas been concentrated in a concentration zone, the polar analyte isdirected into an analytical separation process, for example anelectrokinetic or chromatographic separation process. The concentrationand localization methods of the present invention are particularlyadvantageous where the subsequent analytical separation process iselectrophoresis because the pre-separation concentration step canprovide a concentrated and narrow injection zone leading to bothincreased separation performance, e.g., decreased plate height, andenhanced detectability of the separated components.

In a preferred embodiment of the present invention in which the polaranalyte is a nucleic acid, subsequent to or during the concentration ofthe analyte nucleic acid in the concentration zone, the nucleic acidanalyte is subjected to a nucleic acid hybridization reaction in whichthe concentrated nucleic acid analyte is contacted with one or morecomplementary nucleic acids under conditions suitable forsequence-specific hybridization. In a particularly preferred embodiment,the complementary nucleic acids are bound to a solid support, e.g., anarray of support-bound nucleic acids including one or more potentiallycomplementary nucleic acids. The support-bound nucleic acids may besynthetic polynucleotide probes, cDNA molecules, or any other nucleicacid or nucleic acid analog capable of sequence-specific hybridization.Exemplary arrays of support-bound nucleic acids are described elsewhere,e.g., Singh-Gasson et al., Nature Biotechnology, 17: 974-978 (1999);Blanchard and Friend, Nature Biotechnology, 17: 953 (1999); Brown etal., U.S. Pat. No. 5,807,522. The pre-hybridization concentration stepof the present invention may result in an increased rate ofhybridization, the ability to use a less concentrated sample, or anenhancement of the detectability of the products of the hybridizationreaction. While this embodiment has been described in the context ofnucleic acid hybridization, it will be apparent to one skilled in theart of biochemical instrumentation and analysis that this embodimentcould be equally applied to other process in which a polar analyte iscontacted with a binding complement, e.g., antibody-antigen pairs,receptor-ligand pairs, biotin-avidin pairs, and the like.

In yet another preferred embodiment of the methods of the presentinvention, during or subsequent to the concentration of the polaranalyte in a concentration zone, the polar analyte is detected using adetector, for example a fluorescence detector. In this to embodiment,the pre-detection concentration step can lead to enhanced detectabilityof the polar analyte thereby leading to a more sensitive measurement, orthe opportunity to use a less sophisticated or expensive detectionsystem, e.g., UV absorbence rather than laser-induced fluorescence.Where detection takes place during the concentration process, theconcentration process may be monitored in real time.

In yet another preferred embodiment of the invention, during orsubsequent to the concentration of the polar analyte, the concentratedpolar analyte is contacted with a reactant and a chemical reaction iseffected between the reactant and the concentrated polar analyte. Suchreactions may include chemical, immunological, or enzymatic processes,e.g., analyte labeling, protein digestion, DNA digestion orfragmentation, DNA synthesis, and the like. The concentration step maylead to an increased reaction rate or enhanced detectability of thereaction products.

III. Devices

As is evident based on the foregoing discussion of the various preferredmethods according to the present invention, a wide variety of devicesmay be constructed to carry out the methods. The particular elements,lay-out, dimensions, materials, and experimental conditions used toconstruct and operate the devices of the invention may vary depending onthe particular method or application being addressed.

Generally, a device according to the present invention includes a meansfor generating an alternating electric field and a means for effectingthe relative translation of a polar analyte with respect to thealternating electric field along a translation path. The device isconstructed so that in operation a concentration zone is formed at theintersection of the translation path and the alternating electric field.Several exemplary devices according to the present invention aredescribed below.

FIG. 2A shows a schematic representation of an exemplary microfluidicconcentration and detection device 119 according to the presentinvention. The device 119 comprises an analyte loading reservoir 120, atranslation channel 130, and a waste reservoir 125, such that theanalyte loading reservoir 120 is in fluid communication with the wastereservoir 125 through the translation channel 130. Both the analyteloading reservoir 120 and the waste reservoir 125 contain electrodes 150a and 150 b for effecting the electrokinetic translation of an analytealong the translation channel 130. The electrodes 150 a and 150 b areeach connected to a power supply 170 for providing a voltage differencebetween electrodes 150 a and 150 b and thereby effecting an electricfield along the translation channel 130 sufficient to cause theelectrokinetic translation of an analyte along the translation channel130. The device 119 further comprises a second pair of electrodes 140 aand 140 b located at opposite sides of the translation channel 130 so asto effect an alternating electric field across the width of thetranslation channel 130. The electrodes 140 a and 140 b are connected toan alternating field power supply 160 for providing a time-variantvoltage difference between electrodes 140 a and 140 b and therebyeffecting an alternating electric field substantially across thetranslation channel 130. A concentration zone 181 is located betweenelectrodes 140 a and 140 b. Optionally, the device 119 further includesa detector (not shown) positioned such that material located in theconcentration zone 181 may be detected. In addition, the device 119further optionally includes a computer 180 connected to the power supply170, the alternating field power supply 160, and to the detector (notshown) to control and monitor the operation of the device and to managethe acquisition, analysis, and presentation of data.

FIG. 2B shows a variant of the device described in FIG. 2A 191 in whicha polynucleotide hybridization array 192, or an array of any othersupport-bound binding moiety, is located in the concentration zone 181located between electrodes 140 a and 140 b. The location of thehybridization array 192 is such that an analyte to be contacted with thearray is concentrated in the concentration zone 181 adjacent to thearray prior to hybridization in order to increase the rate of masstransfer of the analyte to the array and enhance the detectability ofthe hybridized analyte.

In operation, the device 191 works generally as follows. The translationchannel 130 and the waste reservoir 125 are filled with a supportingmedium, e.g., a low ionic strength buffered electrolyte solution, and apolar analyte is placed in the analyte loading reservoir 120, e.g.,using a conventional micropipette. Then, power supply 170 is activatedthereby causing the electrokinetic translation of the analyte out of theanalyte loading reservoir 120 and into the translation channel 130.Next, before the analyte reaches the concentration zone 181 locatedgenerally between pair of electrodes 140 a and 140 b, the alternatingfield power supply 160 is activated. As the analyte moves through thetranslation channel 130 into the concentration zone 181 formed by theintersection of the alternating electric field and the translationchannel 130, the polar analyte is trapped and concentrated in theconcentration zone. This process is continued until the desired amountof analyte is located in the concentration zone. Finally, the amount ofpolar analyte in the concentration zone is detected by the detector.Alternatively, the detector monitors the concentration zone throughoutthe process in order to detect the time course of the concentrationprocess. Or, in the device 191 shown in FIG. 2B, the concentrationprocess is continued until the concentration of the polar analyte issufficient to effect the hybridization step.

FIG. 2C shows another variant of the device described in FIG. 2A 192 inwhich a frit 193 is located in the concentration zone 181 locatedbetween electrodes 140 a and 140 b. While the theoretical explanationfor this highly unexpected and beneficial phenomena is not wellunderstood, and the present invention is not intended to be limited inany way by the following or any other theoretical explanation, it isthought by the inventors that the frit 193 creates spatialnonuniformities in the alternating electric field which, in certaincircumstances, serves to enhance the concentration effect of theinvention. The frit 193 has a porous structure comprising through-porescontaining a fluid medium such that material can be transported throughthe frit. Preferably, the pore structure of the frit comprises poreshaving effective internal diameters of between 0.5 and 50 μm.

In one embodiment, the frit is made up of an insulating matrix such thatthe AC electrical conductivity of the insulating matrix is substantiallyless than the AC electrical conductivity of the fluid medium containedin the pores of the frit. Preferably, the electrical conductivity of thefluid medium is 3 times greater than that of the insulating matrix, andmore preferably between 10 and 1000 times greater than that of theinsulating matrix. The insulating matrix may be formed from anyinsulating material that can be fabricated into a frit. Exemplarymaterials include plastics, ceramics, and the like. Preferably, theinsulating matrix is a plastic such as polymethylmethacrylate.

In an alternative embodiment, the frit includes electrically-conductiveparticles suspended in the insulating matrix such that there exists aplurality of electrically isolated regions each containing one or moreconductive particles. In an ideal case, each particle would beelectrically isolated from all other particles by the insulating matrix.The conductive particles may be formed from any conductive orsemi-conductive material, but preferably the particles are metallic,e.g., silver, gold, platinum, copper, and the like, or semiconductormaterials, e.g., gallium arcinide. In a preferred embodiment, theparticles are substantially spherical and have a diameter of betweenabout 0.5 μm to about 50 μm.

Methods for fabricating frits according to the present invention arewell known. Briefly, one exemplary preferred frit fabrication procedureis as follows. Dissolve 1 gram of Plexiglass in 250 ml of chloroform atroom temperature to form a Plexiglass/chloroform mixture. Next, mix 20grams of 150 mesh (99.5%) copper basis copper particles (Alfa AESAR,stock # 10160) with 2 ml of the plexiglass/chloroform mixture to form aparticle suspension. The metal particles should be approximately 2-10 μmin diameter. Mixing should be performed at room temperature by swirlingthe suspension until it becomes evenly mixed for approximately 5minutes. To form the frit, aspirate a 2-5 mm length of the mixture intoa micropipet (Micropipetts calibrated color coded disposable, VWRScientific, cat #53432-921, Size 100 ul) using a conventional pipettebulb. Allow the aspirated suspension to dry partially for approximately5 minutes and then push the suspension farther into the micropipetteusing a rigid wire or rod. Finally, allow the frit to dry for anadditional 30 minutes at room temperature. The frit may then be used insitu or be mechanically removed from the micropipette.

FIG. 3A shows a schematic representation of another exemplarymicrofluidic device 200 according to the present invention. The device200 comprises an analyte loading reservoir 205, an electrokineticseparation channel 225, and a waste reservoir 210, such that the analyteloading reservoir 205 is in fluid communication with the waste reservoir210 through the electrokinetic separation channel 225. The analyteloading reservoir 205 and the waste reservoir 210 contain electrodes 215a and 215 b, respectively, for effecting an electrokinetic translationof a polar analyte along the electrokinetic separation channel 225.Electrodes 215 a and to 215 b are connected to power supply 240. Thedevice further comprises trapping electrodes 220 a and to 220 b foreffecting an alternating electric field between electrodes 220 a and 220b. Electrodes 220 a and to 220 b are connected to alternating-voltagepower supply 230. The device further comprises a detector 245 located ata distal end of the electrokinetic separation channel 225 relative tothe loading reservoir 205 for detecting the polar analyte in a detectionzone 246 after the electrokinetic separation is substantially complete.Optionally, the device 200 includes a computer 235 which is connected topower supply 230, power supply 240, and detector 245, to control andmonitor the operation of the device and to manage acquisition, analysis,and presentation of data. The device 200 may optionally further includea second alternating voltage power supply and pair of electrodes (notshown) located proximate to the detection zone 246 for trapping andconcentrating the separated components of the polar analyte prior to orduring detection in order to further increase the detectability of theseparated components.

In operation, the device 200 works as follows. First, the electrokineticseparation channel 225 and the waste reservoir 210 are filled with anelectrolyte solution capable of supporting the electrokinetictranslation of the polar analyte along the electrokinetic separationchannel 225. Then, the analyte loading reservoir 205 is filled with ananalyte, for example using a conventional micropipette. Next, powersupply 240 is activated in a forward polarity to initiate theelectrokinetic translation of the polar analyte along the electrokineticseparation channel 225. In addition, power supply 230 is activated inorder to establish the alternating electric field used to trap andconcentrate the polar analyte in the concentration zone 181 locatedapproximately between electrodes 220 a and to 220 b. Optionally, toincrease the amount of analyte trapped in the concentration zone, thepolarity of power supply 240 may be cycled between the forward polarityand a reverse polarity such that the polar analyte is translated backand forth through the concentration zone. Once a sufficient amount ofthe polar analyte is located and concentrated in the concentration zone,the power supply 240 is activated in the reverse polarity therebydrawing uncaptured material out of the electrokinetic separation channel225 and back in to loading reservoir 205. At this point, any analyteremaining in analyte loading reservoir 205 may be removed and replacedwith an appropriate electrolyte solution. Next, power supply 230 isturned off and the electrokinetic separation process is continued alongthe electrokinetic separation channel 225 until the components of thepolar analyte are transported to the detection zone 246 and detected bythe detector 245.

FIG. 3B shows a schematic representation of yet another exemplarymicrofluidic device 420 according to the present invention. The device420 is a variant of the device 200 of FIG. 3A in which the singleanalyte loading reservoir 205 in device 200 is replaced with a pair ofreservoirs: an analyte loading reservoir 405 and an analyte wastereservoir 410. The analyte loading reservoir 405 contains electrode 400and the analyte waste reservoir 410 contains electrode 415. Analyteloading reservoir 405 is connected to the electrokinetic separationchannel 225 by branch channel 406, and analyte waste reservoir 410 isconnected to electrokinetic separation channel 225 by branch channel407. Other elements of the device 420 are as described with respect todevice 200 in FIG. 3A.

In operation, the device 420 works as follows. First, the electrokineticseparation channel 225, the waste reservoir 210, the branch channels 407and 406, and the analyte waste reservoir 410 are filled with anelectrolyte solution capable of supporting the electrokinetictranslation of the polar analyte along the electrokinetic separationchannel 225. Then, the analyte loading reservoir 205 is filled with ananalyte, for example using a conventional micropipette. Next, powersupply 240 is activated in a forward polarity to effect a potentialdifference between electrodes 400 and 215 b, to initiate theelectrokinetic translation of the polar analyte through branch channel406 and along the electrokinetic separation channel 225 and intoconcentration zone 181. As before, power supply 230 is activated inorder to establish the alternating electric field used to trap andconcentrate the polar analyte in the concentration zone 181 locatedsubstantially between electrodes 220 a and to 220 b. Optionally, thepolarity of power supply 240 may be cycled between the forward polarityand a reverse polarity such that the polar analyte is translated backand forth through the concentration zone. Once a sufficient amount ofthe polar analyte is located and concentrated in the concentration zone,the power supply 240 is activated in the reverse polarity to effect apotential difference between electrodes 415, 400 and 215 b such thatuncaptured material is drawn out of the electrokinetic separationchannel 225 and the analyte loading reservoir 405 and into the analytewaste reservoir 410. Optionally, after sweeping the uncaptured analyteinto analyte waste reservoir 410, analyte present in waste reservoir 415may be removed and replaced with an appropriate electrolyte solution.Next, power supply 230 is turned off and the electrokinetic separationprocess is continued along the electrokinetic separation channel 225between electrodes 400 and 215 b until the components of the polaranalyte are transported to the detection zone 246 and detected by thedetector 245.

The fluidic channels used in many of the preferred devices of theinvention may be any channel capable of supporting a sample containing apolar analyte and solvent or other supporting media required to carryout a method of the invention. The channels may be discrete, for exampleindividual capillary tubes, or formed as part of an integratedmicrofluidic device, e.g., channels etched in a glass substrate.Preferably, the fluidic channels are formed as part of an integratedmicrofluidic device including one or more intersecting channels andreservoirs. Exemplary microfluidic devices, and several alternativemethods for device fabrication, are disclosed in Soane and Soane, U.S.Pat. Nos. 5,750,015 and 5,126,022; Manz, U.S. Pat. Nos. 5,296,114 and5,180,480; Junkichi et al., U.S. Pat. No. 5,132,012; and Pace, U.S. Pat.No. 4,908,112. Other references describing microfluidic devices includeHarrison et al., Science, 261: 895 (1992); Jacobsen et al., Anal. Chem.66: 2949 (1994); Effenhauser et al., Anal. Chem. 66:2949 (1994); andWoolley and Mathies, P.N.A.S. USA, 91:11348 (1994). A general discussionof microfabrication techniques is provided by Madou in Fundamentals ofMicrofabrication, CRC Press, Boca Raton, Fla. (1997).

The fluidic channels may be present in the device in a variety ofconfigurations, depending on the particular application being addressed.The internal volume of a channel will preferably range from about 1 nlto about 10 μl, and more preferably from about 10 nl to about 2 μl. Thelength of a channel will generally range from about 1 mm to about 50 cm,usually between about 5 cm to 30 cm. However, in certain applications,channels may have a length up to or greater than 100 cm, e.g., where thechannel is used as an electrokinetic or chromatographic separationchannel. The cross-sectional dimensions (e.g., width, height, diameter)will range from about 1 μm to about 400 μm, usually from about 20 μm toabout 200 μm. The cross-sectional shape of the channel may be anycross-sectional shape, including but not limited to circular, ellipsoid,rectangular, trapezoidal, square, or combinations of the foregoingshapes. The fluidic channels may be straight, serpentine, helical,spiral, or any other configuration, depending on the requirements of aparticular application. For example, if it is desired to construct along fluidic channel in a small substrate, it may be advantageous toconstruct a channel having a spiral or serpentine shape.

The fluidic channels of the device may optionally comprise, and usuallywill comprise, fluid reservoirs at one or both termini, i.e., eitherend, of the channels. Where reservoirs are provided, they may serve avariety of purposes including a means for introducing various fluidsinto the channel, e.g., buffer, elution solvent, sieving media, reagent,rinse or wash solutions; means for receiving waste fluid from a channel,e.g., an electrokinetic flowpath; or, as electrode reservoirs forcontacting an electrode with an electrolyte and for supplying ions tosupport an electrokinetic process. Generally, the reservoirs will have avolume of between 1 μl and 100 μl, preferably between 1 μl about 1 μland 10 μl. Larger reservoirs may be desirable if the reservoirs servemultiple fluidic channels.

The subject devices may also optionally comprise an interface system forassisting in the introduction of an analyte into the device. Forexample, where the analyte is to be introduced into the device using asyringe, the interface system may comprise a syringe interface whichserves as a guide for the syringe needle into the device, e.g., as aseal over an analyte introduction reservoir.

Depending on the particular application, configuration, and materialsfrom which the device is fabricated, a detection region for detectingthe presence of a particular analyte may be included in the device,e.g., element 246 in FIG. 3. For example, preferably at least one regionof the electrokinetic channel includes a detection region that isfabricated from a material that is optically transparent, generallyallowing light of wavelengths ranging from 180 to 1500 nm, usually 250to 800 nm, to be transmitted through the material with low transmissionlosses, i.e., less than about 20%, preferably less than about 5%.Suitable optically transparent materials include fused silica, certainoptically-clear plastics, quartz glass, borosilicate glass, and thelike.

The subject devices according to the present invention may be fabricatedfrom a wide variety of materials, including glass, fused silica,acrylics, thermoplastics, silicon, and the like. Preferably, thematerials will have high dielectric breakdown potential, e.g., greaterthan about 100 kV/cm, be mechanically rigid, be chemically compatiblewith the polar analyte and any associated solvents or media, and have alow dielectric loss factor, e.g., less than about 0.05 at 1 MHz. Thevarious components of the integrated device may be fabricated from thesame or different materials, depending on the particular use of thedevice, the economic concerns, solvent compatibility, optical claritycolor, mechanical strength, mechanical features, electrical properties,thermal properties, and the like. For example, a planar substratecomprising microfluidic flowpaths and a cover plate may be fabricatedfrom the same material, e.g., polymethylmethacrylate (PMMA), ordifferent materials, e.g., a substrate of PMMA and a cover plate ofglass. For applications where it is desired to have a disposableintegrated device, due to ease of manufacture and cost of materials, thedevice will typically be fabricated from a plastic. For ease ofdetection and fabrication, the entire device may be fabricated from aplastic material that is optically transparent with respect to theoptical wavelengths used for detection. Also of interest in certainapplications are plastics having low surface charge under conditions ofelectrophoresis. Particular plastics useful for the fabrication ofdisposable devices according to the present invention include but arenot limited to polymethylmethacrylate, polycarbonate, polyethyleneterepthalate, polystyrene or styrene copolymers.

Optionally, the surface properties of materials used to fabricate thedevices may be altered in order to control analyte-wall interactions,electroendoosmosis, bonding properties, or any other surface-mediatedproperty of the materials. The surface modifications may be based oncovalent attachment of coating agents, or physical attachment, e.g., byionic, hydrophobic, or van der Waals interactions. Exemplary surfacemodification techniques are described elsewhere, e.g., Hjerten, U.S.Pat. No. 4,680,201; Cobb et al., Anal. Chem., 62: 2478-2483 (1990); vanAlstine et al., U.S. Pat. No. 4,690,749; and Belder and Schomburg,Journal of High Resolution Chromatography, 15: 686-693 (1992):

The devices according to the present invention may be fabricated usingany convenient means used for fabricating like devices. In a preferredembodiment of the invention, conventional molding and casting techniquesare used to fabricate the devices. For example, for devices preparedfrom a plastic material, a silicon mold master which is a negative forthe channel structure in the planar substrate of the device can beprepared by etching or laser micromachining. In addition to having araised ridge which will form the channel in the substrate, the silicamold may have a raised area which will provide for a cavity into theplanar substrate for forming reservoirs or other fluidic features. Whereconvenient, the procedures described in U.S. Pat. No. 5,110,514 may beemployed. A cover plate may be sealed to the substrate using anyconvenient means, including ultrasonic welding, adhesives, etc.

Alternatively, the devices may be fabricated using photolithographictechniques as employed in the production of microelectronic computerchips as follows. First, a substrate support such as apolymethylmethacrylate card approximately the size of a conventionalcredit card is provided. The surface of the card itself is notelectrically conducting nor is the card. On the card is first depositeda thin layer of an electrically conducting material, e.g., a metal. Thecoating may be applied by a variety of different techniques known tothose skilled in the art and may be comprised of a variety of differenttypes of materials provided they are capable of conducting electricityand preferably chemically inert, e.g., platinum, gold, and the like. Thelayer is preferably thin; on the order of 100 angstroms to a few micronsof thickness. The electrically conducting layer is then coated with alayer of material which is both light-sensitive and non-conducting. Oncethe light-sensitive, non-conducting layer completely covers theelectrically conducting layer, a mask is applied to the surface of thelight-sensitive, non-conducting layer. After the mask covers the layer,it is exposed to light resulting in a pattern of portions of thelight-sensitive material being solvent soluble and portions beingsolvent insoluble. The soluble portions are washed away and the exposedelectrically conducting material etched away leaving traces of wires andconnectors to the wires under the insoluble portion of the lightsensitive material. The underlying electrically conducting material willprovide electrode connections to the fluidic channels and reservoirs. Byremoving a portion of the insoluble material from the ends or connectorsof the electrically conducting traces remaining from the electricallyconducting layer, electrical connection can be made with the electrodeconnectors to the fluidic elements. In addition the electrode traces areprotected from wear and abrasion by the protective coating. As will beapparent to those skilled in the art, the mask utilized in the aboveproduction procedure can be produced so as to provide essentially anunlimited number of different electrode connections to the device, e.g.,see S. M. Sze, VLSI Technology, Second Edition, McGraw-Hill, New York,N.Y. (1988).

Where the devices are formed from glass, standard etching process may beused, e.g., K. Fluri et al., Anal. Chem., 66: 4285-4290 (1996); Z. Fanand D. J. Harrison, Anal. Chem., 66: 177-184 (1994).

Alternatively, rather than utilizing the photolithographic or moldingand casting techniques generally described above to fabricate thedevices of the present invention, it is possible to utilize otherfabrication techniques such as employing various types of lasertechnologies and/or other technologies such as silk-screening and vapordeposition which make it possible to provide extremely small (in size)and large numbers of electrodes and fluidic channels, or lamination, orextrusion techniques.

Generally, the fluidic channels will contain a supporting medium tosupport the analyte. The medium may be an organic solvent, a buffersolution, a polymeric solution, a surfactant micellular dispersion, orgel of the type generally used in connection with biochemical separationtechniques. Preferably, the medium will have a low ionic strength, e.g.,below about 100 mM salt, more preferably below about 10 mM salt, andcomprise a solvent having a low dielectric constant, e.g., below about80. A particularly preferred medium comprises an entangled aqueouspolymer solution, e.g., Madabhushi et al, U.S. Pat. No. 5,567,292.

The electrodes used in the devices of the invention, e.g., to effect thealternating electric field, may be fabricated using conventional methodsfrom conventional materials. Exemplary methods for electrode fabricationinclude photolithography, silkscreen techniques, and a simple wireelectrode. Preferably, the materials used to form the electrodes areelectrically conductive and chemically inert. Particularly preferredmaterials include gold, platinum, tungsten, and the like.

Preferably the electrodes used to effect the alternating electric fieldhave a shape which serves to form an electric field that results in asingle, well-defined concentration zone having the desired dimensions.FIG. 4 shows several exemplary electrode geometries; 300, 305, 310, 315,and 320. The spacing of the electrodes is chosen so as to generate anelectric field having sufficient strength to trap a polar analyte ofinterest but not so high as to cause excessive bubble formation at theelectrodes, e.g., see Washizu et al., IEEE Transactions on IndustryApplications, 30 (4): 835-843 (1994). Generally, the spacing between theelectrodes is preferably between about 50 μm and 2 mm.

Preferably, the electrodes used to effect the alternating electric fielddo not protrude into the translation path or otherwise come into directphysical contact with the translation path. Rather, the electrodes arepreferably positioned such that the alternating electric field producedbetween the electrodes intersects the translation path, but theelectrodes themselves are physically removed from the translation path,e.g., using an isolation layer or an electrical connection through aconductive bridge. Exemplary materials useful for forming an isolationlayer include SiO₂, Al₂O₃, polyimide, diamond, glass, and the like.Isolating the electrodes from the translation path is preferred becauseit serves to minimize the extent to which the electrodes may becomefouled by the analytes or any other material present in the translationpath, or become pasiviated due to oxidation or other chemicaltransformation of the electrode surface.

The means for effecting an alternating electric field may include anyconventional alternating current power supply using conventionalelectrical connectors. An exemplary alternating current power supplysuitable for use in the present invention is a Model 33120A 15 MHzFunction/Arbitrary Waveform Generator function generator from AgilentTechnologies. The means for effecting an alternating electric fieldfurther includes the above-described electrodes and electricalconnections between the electrodes and the alternating current powersupply.

The electrodes may be fabricated and integrated into the devices of theinvention using any one of a number of conventional techniques. Forexample, wire electrodes may be mechanically attached to the device,electrodes may be incorporated into the device during a lamination stepor during a casting step, electrodes may be deposited onto the deviceusing plasma deposition or electrochemical deposition techniques.

As discussed above, certain embodiments of the present invention includea detection system for detecting an analyte during or subsequent to ananalysis. Any sort of conventional detection system may be used with thepresent invention, including systems for measuring fluorescence,radioactivity, optical absorbence, fluorescence polarization, electricalconductivity, electrochemical properties, refractive index, and thelike. A particularly preferred detection system employs laser-excitedfluorescence.

In a preferred embodiment of the present invention, the channels,reservoirs, or concentration zone of the microfluidic device aremaintained at a controlled temperature using a temperature controlsystem. For example, temperature control may be desirable in order toincrease the reproducibility of an analysis, control the properties of amedium or analyte, or to speed up an analysis; A preferred temperaturecontrol system may include a heating element, e.g., a resistive heatingelement in combination with a fan, a cooling device, e.g., a Peltierdevice or other conventional refrigeration device, an enclosedthermally-isolated chamber, one or more temperature measurement sensors,and a programmable feedback control system. Preferably, the temperaturecontrol system is connected to a computer for controlling and monitoringthe overall system.

The devices of the present invention may be used in combination withrobotic systems to automate certain steps of a process, e.g., a robotmay be used to manipulate a plurality of microfluidic devices forhigh-throughput multi-device applications, or to introduce an analyteinto a device of the invention, or to transfer a medium into or out ofthe device. Such robots may be any kind of conventional laboratoryrobot, preferably with fluid-handling capability, e.g., a Beckman BioMeksystem.

In certain embodiments of the present invention, a computer is used tomonitor or control various aspects of the device including: controllingthe operation of the components of the device, e.g., power supplies,temperature controller, pumps, etc.; controlling systems associated withthe device, e.g., laboratory robots; monitoring the performance of thedevice, e.g., measuring electric field strengths, temperatures, orpressures; managing of data acquisition, data analysis, and datapresentation activities; or providing a convenient user interface forthe programmable operation of the device. The computer of the presentinvention may be any type of conventional programmable electroniccomputer, e.g., a personal computer. The computer may be connected toother elements of a system through conventional devices, e.g., an A/Dconverter.

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference to the same extent as if eachindividual publication, patent, or patent application was specificallyand individually indicated to be incorporated by reference.

Although only a few embodiments have been described in detail above,those having ordinary skill in the art of chemical instrumentation willclearly understand that many modifications are possible in the preferredembodiments without departing from the teachings thereof. All suchmodifications are intended to be encompassed within the scope of thefollowing claims.

1-34. (canceled)
 35. A device for manipulating a plurality of cells,comprising: (a) a translation channel; (b) at least one electrodeconfigured to provide an alternating electric field intersecting thetranslation channel, wherein the alternating electric field issufficient to trap and concentrate at least a portion of the cellslocated in a concentration zone formed by the intersection of thetranslation channel and the alternating electric field; and (c) a meansfor effecting translation of the cells along the translation channelsuch that at least a portion of the cells pass through the concentrationzone.
 36. The device of claim 35, wherein the means for effectingtranslation of the cells comprises a gravitational force.
 37. The deviceof claim 35, wherein the means for effecting translation of the cellscomprises a centrifugal force.
 38. The device of claim 35, wherein themeans for effecting translation of the cells comprises an electrokineticforce.
 39. The device of claim 38, wherein the electrokinetic forcecomprises electrophoresis.
 40. The device of claim 39, wherein theelectrophoresis is effected by a direct current applied to a firstelectrode and a second electrode, wherein the first electrode is locatedtoward a first end of the translation channel and the second electrodeis located toward a second end of the translation channel.
 41. Thedevice of claim 35, wherein the means for effecting translation of thecells comprises a magnetic force.
 42. The device of claim 35, whereinthe means for effecting translation of the cells comprises a hydraulicforce.
 43. The device of claim 35, wherein the means for effectingtranslation of the cells comprises a capillary force.
 44. The device ofclaim 35, further comprising: a detector positioned to detect cells inthe concentration zone; and a temperature control system in thermalcommunication with the concentration zone.
 45. A device for manipulatinga plurality of cells, comprising: (a) an elongated channel including (i)first and second end regions, and (ii) first and second lateral sideregions; (b) a first set of electrodes, wherein at least one electrodeof said first set is disposed toward the first end region, and at leastone other electrode of said first set is disposed toward the second endregion; and (c) a second set of electrodes, wherein at least oneelectrode of said second set is disposed toward the first lateral sideregion, and at least one other electrode of said second set is disposedtoward the second lateral side region.
 46. The device of claim 45,further comprising a first power supply disposed for electricalcommunication with said first set of electrodes and a second powersupply disposed for electrical communication with said second set ofelectrodes.
 47. The device of claim 46, further comprising a computerdisposed for communication with said device, said computer beingoperable to establish a translational electrical field lengthwise alongat least a portion of said elongated channel and a trapping electricalfield transverse to said translational electrical field, with saidtranslational and trapping electrical fields crossing one another at anintersection defining a concentration zone.
 48. The device of claim 45,further comprising a detector positioned to detect cells in theconcentration zone.
 49. The device of claim 47, further comprising adetector positioned to detect cells in the concentration zone, whereinthe computer is configured to alter the translational electric field inresponse to data received from the detector.
 50. The device of claim 47,further comprising a detector positioned to detect cells in theconcentration zone, wherein the computer is configured to alter thetrapping electric field in response to data received from the detector.51. The device of claim 45, further comprising a temperature controlsystem.
 52. The device of claim 51, wherein the temperature controlsystem is in thermal communication with the concentration zone.
 53. Thedevice of claim 47, further comprising a temperature control system,wherein the computer is disposed for operable communication with thetemperature control system.
 54. A method of manipulating a plurality ofcells, comprising: (a) effecting the relative translation of the cellsalong a translation path; and (b) applying an alternating electric fieldtransverse to said translation path and intersecting said translationpath, such that at least a portion of the cells are trapped andconcentrated in a concentration zone formed by the intersection of thealternating electric field and the translation path.
 55. The method ofclaim 54, wherein effecting the relative translation of the cellscomprises applying at least one of a gravitational force, a centrifugalforce, an electrokinetic force, a magnetic force, a hydraulic force, ora capillary force.
 56. The method of claim 54, wherein effecting therelative translation of the cells comprises applying a magnetic force.57. The method of claim 54, wherein effecting the relative translationof the cells comprises applying an electrokinetic force.
 58. The methodof claim 57, wherein the electrokinetic force comprises electrophoresis.59. The method of claim 58, wherein the electrophoresis is effected by adirect current applied to a first electrode and a second electrode,wherein the first electrode is located toward a first end of thetranslation channel and the second electrode is located toward a secondend of the translation channel.
 60. The method of claim 54, wherein atime-vs.-field strength profile of the alternating electric field isrectangular.
 61. The method of claim 54, wherein the time-averagedintegrated field strength of the alternating electric field taken overone complete cycle is zero.
 62. The method of claim 54, wherein thefrequency of the alternating electric field is between about 10 Hz and100 MHz.
 63. The method of claim 62, wherein the frequency ofalternating electric field is between about 1 KHz and 100 KHz.
 64. Themethod of claim 54, wherein the maximum field strength of thealternating electric field is between 100 V/cm and 100,000 V/cm.
 65. Themethod of claim 64, wherein the maximum field strength of thealternating electric is between 1,000 V/cm and 20,000 V/cm.
 66. Themethod of claim 54, wherein the alternating electric field is spatiallynon-uniform.
 67. The method of claim 54, further comprising detecting atleast one cell before, during, or after concentration.
 68. The method ofclaim 54, further comprising releasing at least a portion of the cellsfrom the concentration zone.
 69. The method of claim 68, furthercomprising detecting at least one cell after the releasing.
 70. A devicefor manipulating a plurality of cells, comprising: (a) a translationchannel; (b) one or more electrodes located to provide an alternatingelectric field intersecting the translation channel, wherein thealternating electric field is sufficient to trap and concentrate atleast a portion of the cells located in a concentration zone formed bythe intersection of the translation channel and the alternating electricfield; (c) a means for effecting translation of the cells along thetranslation channel such that at least a portion of the cells passthrough the concentration zone; and (d) an array of support-boundbinding complements located in the concentration zone.
 71. The device ofclaim 70, wherein the binding complements are selected from antibodiesand receptor ligands.
 72. The device of claim 70, further comprising adetector positioned to detect cells located in the concentration zone.73. The device of claim 72, wherein the detector is configured to detecteach location in the array.
 74. A device for manipulating a plurality ofcells, comprising: (a) a translation channel; (b) one or more electrodeslocated to provide an alternating electric field intersecting thetranslation channel, wherein the alternating electric field issufficient to trap and concentrate at least a portion of the cellslocated in a concentration zone formed by the intersection of thetranslation channel and the alternating electric field; (c) a means foreffecting translation of the cells along the translation channel suchthat at least a portion of the cells pass through the concentrationzone; and (d) a frit located in the concentration zone, wherein the fritis made of an insulating matrix.
 75. The device of claim 74, wherein theAC electrical conductivity of the insulating matrix is less than the ACelectrical conductivity of a fluid medium located in pores of the frit.76. The device of claim 74, wherein the AC electrical conductivity ofthe insulating matrix is at least 3 times less than that of the fluidmedium.
 77. The device of claim 76, wherein the AC electricalconductivity of the insulating matrix is between about 10 and 1000 timesless than that of the fluid medium.